30 Diagnostic Cytopathology, Vol 42, No 1 VC 2013 WILEY PERIODICALS, INC.

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1 Cytopathologic Diagnosis on Joint Lavage Fluid for Patients with Temporomandibular Joint Disorders Toshinari Mikami, D.D.S., Ph.D., 1* Akiko Kumagai, D.D.S., Ph.D., 2 Tomoyuki Aomura, D.D.S., Ph.D., 2 Fawad Javed, D.D.S., Ph.D., 3 Yoshiki Sugiyama, D.D.S., Ph.D., 2 Harumi Mizuki, D.D.S., Ph.D., 2 and Yasunori Takeda, D.D.S., Ph.D. 1 Temporomandibular joint (TMJ) disorders (TMD) are usually diagnosed based on the patient s clinical findings and the results of image investigations; however, understanding of the inflammatory process in TMJ is difficult. In addition, many of the TMJ disease types share common principal symptoms. Therefore, TMJ diseases in the early stage can be misdiagnosed with TMD. It is hypothesized that cytopathologic examination of the joint lavage fluids is useful in interpreting the TMD-associated inflammatory process from a cellular aspect. The aim of this study was to assess the TMJ lavage fluid cytopathologically in TMD patients. Thirty-nine patients, clinically diagnosed as TMD, were included in the present study. Clinical symptoms of the patients were recorded. Forty-four samples of TMJ lavage fluid were collected and paraffin-embedded cell sections were made by cell block tissue array method. Cytologic conditions in upper articular cavity of TMJ were cytopathologically diagnosed and were compared with the clinical symptoms of each 1 Division of Anatomical and Cellular Pathology, Department of Pathology, Iwate Medical University, Nishitokuta Yahaba, Shiwagun Iwate, , Japan 2 Division of Maxillofacial Surgery, Department of Oral and Maxillofacial Surgery, School of Dentistry, Iwate Medical University, 19-1 Uchimaru, Morioka Iwate, , Japan 3 Engineer Abdullah Bugshan Research Chair for Growth Factors and Bone Regeneration, 3D Imaging and Biomechanical Laboratory, College of Applied Medical Sciences, King Saud University, Riyadh, 11545, Saudi Arabia *Correspondence to: Toshinari Mikami, Division of Anatomical and Cellular Pathology, Department of Pathology, Iwate Medical University, Nishitokuta Yahaba, Shiwa-gun Iwate , Japan. toshi_m@sea.plala.or.jp Contract grant sponsor: JSPS KAKENHI; contract grant number: Contract grant sponsor: Strategic Medical Science Research Center, Ministry of Education, Culture, Sports, Science and Technology of Japan. Received 19 December 2012; revised 21 March 2013; Accepted 3 June 2013 DOI: /dc Published online 1 August 2013 in Wiley Online Library (wileyonlinelibrary.com). patient. Cell components were detected in 22 of the 44 analyzed joint lavage fluids. There was a correlation between cytopathologic findings and clinical symptoms. Variety of cytopathology and inflammatory conditions in patients with similar clinical symptoms were also found. The results suggested that cytopathologic examination of the joint lavage fluids from TMD patients is helpful for gaining an understanding of the inner local conditions of TMJ at the cellular level. Diagn. Cytopathol. 2014;42: VC 2013 Wiley Periodicals, Inc. Key Words: joint lavage fluid; temporomandibular joint disorder; cytopathology; cytodiagnosis; inflammation Temporomandibular joint (TMJ) disorders (TMD) are diagnosed based on the patient s clinical findings and the results of image investigations; however, understanding of the inflammatory processes in TMJ is difficult. In addition, many TMJ disease types (such as crystalline arthritis, 1 12 synovial chondromatosis, and TMD 17,18 ) share some principal symptoms, such as TMJ sounds and pain, lateral deviation, and difficulty in mouth opening. Therefore, TMJ diseases in the early stage can be misdiagnosed with TMD. 15 TMJ arthrocentesis and lavage was first described by Nitzan et al. 19 It is regarded as an effective and less invasive technique to treat acute or chronic closed lock of the TMJ TMJ arthrocentesis and lavage with or without manipulation is often performed early in the treatment of TMD patients to unlock the TMJ closed lock or to remove inflammatory substances from the upper articular cavity. Although the joint lavage fluid is routinely disposed, cytopathologic examination of this fluid may reveal useful information regarding the patient s symptoms at the cellular level. 30 Diagnostic Cytopathology, Vol 42, No 1 VC 2013 WILEY PERIODICALS, INC.

2 CYTOPATHOLOGIC DIAGNOSIS FOR TMD From a pathologic perspective, predominant infiltration of neutrophils in a tissue indicates acute inflammation, and also suggests that the irritant likely causing the pain is still present. The predominant infiltration of lymphocytes and/or plasma cells indicates chronic inflammation, and suggests that at least a few days has progressed since irritant first appeared. Neutrophils and lymphocytes are both predominant in tissues in the transitional phase between acute and chronic inflammation. Presence of monocyte lineage cells or multinucleated giant cells in inflamed tissue suggest the presence of calcium phosphate crystals, which cause crystalline arthritis. 1 3,5,8 12 Furthermore, presence of osteoclasts suggests modeling or remodeling of the TMJ, and the presence of cartilaginous components suggests synovial chondromatosis. 14,15 Correlations between the presence of inflammatory cytokines in the joint lavage fluid of TMD patients and the clinical outcome have been reported It is hypothesized that cytopathologic examination of the joint lavage fluids is useful in interpreting the TMD-associated inflammatory process from a cellular aspect. The aim of the present study was to assess the TMJ lavage fluid in TMD patients using cytopathology. Patients and Methods Ethical Guidelines This study was approved by the research ethics committee of the School of Dentistry, Iwate Medical University, Morioka, Japan, and was performed in adherence with the guidelines of the Declaration of Helsinki. The study participants were selected from patients receiving treatments of pumping manipulation or joint lavage at the School of Dentistry, Iwate Medical University, Morioka, Japan. An information sheet that explained the objectives and methods of the present investigation was distributed to the TMD patients, and volunteering individuals were presented a consent form. It was mandatory for the study participants to have read and signed the consent form prior to inclusion in the present investigation. The study was conducted between June 2008 and May Study Participants In total, 39 patients (7 male and 32 female) previously clinically diagnosed with TMD volunteered to participate in the present investigation. The median age was 50 years (range of 18 72), and the mean age was 48.5-years-old. In the present study, patients clinically diagnosed exclusively with TMJ disorder were included. Cell Block Tissue Array Method The cell block tissue array method 31 was used to make paraffin-embedded cell sections. After treatments of pumping manipulation or joint lavage, aspirated joint lavage fluid from the upper articular cavity was placed directly into CytoRich RED Preservative Fluid (BD, Franklin Lakes, NJ) for fixation. The fluid was centrifuged at 2000 rpm for 5 min, and the supernatant was removed. The precipitate was resuspended in 1 ml of 1% alginate sodium. After incubating at room temperature for 10 min, the sample was centrifuged at 2000 rpm for 5 min and the supernatant of alginate sodium was removed. For gelatinization, 20 ll of 1M calcium chloride was added to the precipitate. The gelatinized cell block was placed in a cassette, dehydrated using ethanol, infiltrated with paraffin, and prepared into paraffin-embedded cell blocks. Four micrometer-thick sections were prepared for hematoxylin eosin (HE) staining and immunocytochemistry. Immunocytochemistry Immunocytochemical analyses were performed using the ChemMate EnVision Detection Kit/HRP (DAB) Rabbit/ Mouse (Dako, Glostrup, Denmark) to determine the monocyte lineage cells (macrophages, osteoclasts, or monocyte lineage multinucleated giant cells). For antigen retrieval of CD68, the sections were placed in a microwave for 30 min in 10 mm citrate buffer (ph 6.0). Endogenous peroxidase activity in the sections was blocked with Dako REAL Peroxidase-Blocking Solution (S2023; Dako). We used mouse anti-cd68 monoclonal antibody (1:2, ; Nichirei, Tokyo, Japan), alpha 1- antitrypsin (1:2, ; Nichirei) and alpha 1-antichymotrypsin (1:100, A0022; Dako) as the primary antibody. Positive signals were visualized with 3,3 0 -diaminobenzidine using DakoCytomation Liquid DAB 1 Substrate Chromogen System (K3467; Dako). Results Summary of the patients background and results of cytopathologic analyses of their joint lavage fluid are shown in Table I. Cytopathologic Findings Cell components, such as neutrophils, lymphocytes, plasma cells, eosinophils, macrophages, monocyte lineage multinucleated giant cells (osteoclasts or foreign bodytype giant cells), or cartilaginous tissues were detected in 22 of the 44 analyzed joint lavage fluids. Neutrophils were found in 17 cases, lymphocytes in 16 cases, plasma cells in one case, eosinophils in one case, macrophages in one case, monocyte lineage multinucleated giant cells in two cases, and partially calcified cartilaginous tissues in Diagnostic Cytopathology, Vol 42, No 1 31

3 MIKAMI ET AL. Table I. Summary of Clinical Findings, Cytopathology and Cytopathologic Condition of Joint Lavage Fluids from Patients with TMD Pt no. Gender Age History Pain JS MD MOD Treatments Cytopathology Cytopathologic condition 1 F 18 3 years mm Splint n.d. 2 F 18 1 year mm 2 n.d. 3 F 18 2 years mm 2 Neutrophil, lymphocyte Acute inflammation 4 F 28 1 year Medication Neutrophil Acute inflammation 5 F 31 1 year mm Splint n.d. 6 F years mm Splint Neutrophil, lymphocyte Acute inflammation 7 F 37 8 years mm Splint n.d. 8 M 37 1 day mm Manipulation n.d. 9 F 38 1 year mm Splint Lymphocyte Chronic inflammation, 10 F 38 8 months mm Medicatioin Neutrophil, lymphocyte, Acute/chronic inflammation plasma cell 11 F 38 9 months n.d. 12 F years mm Splint Neutrophil, lymphocyte Acute inflammation 13 F 44 6 months mm Splint Lymphocyte Chronic inflammation 14 F years mm Splint Neutrophil, lymphocyte, macrophage, monocyte Acute/chronic inflammatioin, osteoarthrosis suspect lineage multinucleated giant cell 15 F 46 5 years mm Splint Neutrophil, lymphocyte Acute inflammation 16 M 46 1 month n.d. 17 F years mm Splint n.d. 18 M 48 Unclear n.d. 19 F 50 Unclear mm 2 Neutrophil Acute inflammation 20 F 50 1 year mm Medication n.d. 20 F 50 1 year Splint, pumping n.d. manipulation 21 F months mm Splint Neutrophil, lymphocyte Acute inflammation 22 F 52 6 months mm 2 Neutrophil, lymphocyte Acute inflammation 23 F 55 6 months Splint n.d. 24 F 56 5 months Splint Lymphocyte Chronic inflammation 25 F 57 5 years mm Splint Neutrophil, lymphocyte Acute inflammation 26 F 57 4 months mm Manipulation n.d. 27 F 58 4 months mm 2 n.d. 28 F 60 2 years mm Splint n.d. 29 F 60 3 months mm Splint Neutrophil, lymphocyte, eosinophil, monocyte lineage multinucleated giant cell 29 F 60 4 months mm Medication, splint, joint lavage Neutrophil, lymphocyte 29 F 60 6 months mm Medication, splint, joint neutrophil, lymphocyte, lavage eosinophil 30 M 63 2 years Pumping manipulation n.d. 31 F 30 6 months mm Medication, manipulation Partially calcified cartilage surrounded by fibrous tissue Acute inflammation, remodeling suspect Acute inflammation Acute inflammation Synovial chondromatosis 32 F 64 2 months n.d. 33 F 64 1 year Splint n.d. 34 M years mm Splint Neutrophil, lymphocyte Acute inflammation 35 F months n.d. 36 F 68 5 months mm 2 n.d. 36 F 68 6 months mm Joint lavage Neutrophil Acute inflammation 37 M 70 3 months n.d. 38 M 72 9 months mm Pumping manipulation Neutrophil Acute inflammation 39 F 72 2 months n.d. 39 F 72 3 months mm Pumping manipulation Neutrophil, lymphocyte Acute inflammation JS, joint sound; MD, mandibular deviation; MOD, mouth opening difficulty; n.d., not detected. one case. There were 13 cases in which neutrophils and lymphocytes were present together (Fig. 1). Multinucleated giant cells were detected in patients no. 14 and 29, which were positive for monocyte lineage cell markers (CD68, alpha 1-antitrypsin and alpha 1-antichymotrypsin) (Figs. 2A D). The case of patient no. 31 has been reported previously by the authors as a case report of phase III synovial chondromatosis. 15 Tissue from the lavage fluid consisted of many small islands surrounded by perichondrium, with partial calcification (Fig. 3). It was also positive for toluidine blue, aggrecan, collagen type II, and BMP-2/BMP-4. To summarize the cytopathologic diagnoses, there were 16 cases of acute inflammation, three cases of chronic 32 Diagnostic Cytopathology, Vol 42, No 1

4 CYTOPATHOLOGIC DIAGNOSIS FOR TMD Repeatability of the Examinations and Consistency Between Clinical and Cytopathologic Findings in a Patient Patient no. 29 received a joint lavage three times in three months to remove inflammatory substances in the upper articular cavity because splint therapy and medication did not improve her symptoms. During the three months of treatment, the results of all three cytopathologic analyses were consistent with the clinical symptoms of the patients, showing predominance of neutrophils and lymphocytes. Fig. 1. HE-staining of joint lavage fluid from patient no. 29 at her first visit. Various kinds of inflammatory cells were observed in cell specimens made with cell block tissue array method. (i) Neutrophils, (ii) Lymphocytes, and (iii) Eosinophils (Bar: 50 lm). inflammation, two cases of transition phase between acute and chronic inflammation, one case of suspected arthrosis deformans (based on cytopathology and image investigations), one case of suspected TMJ remodeling (based on cytopathology and image investigations), and one case of synovial chondromatosis. Correlation Between Clinical and Cytopathologic Findings Patients expressed feeling pain in the TMJ area in 36 of the 44 cases. In all of the 22 cases in which cell components were detected, the patients expressed feeling pain. There were 14 cases in which inflammatory cells were not detected, despite the patients feeling pain. Additionally, there were no inflammatory cells detected in the eight cases in which the patients did not feel pain. Monocyte lineage cells (macrophages, osteoclasts, or foreign body-type giant cells) were detected in the samples from patients no. 14 and 29; however, in both cases, crystalline materials were not detected via optical microscopy and polarizing microscopy. Panoramic radiograph of patient no.14 showed destructive changes of the bilateral condyle. Analysis by cone beam computed tomography revealed an erosion of the bilateral condyle head and right articular fossa, and a formation of a bone spur on the right condyle head (Fig. 4). The destructive change was more evident on the right side of TMJ in both image investigations. The clinical diagnosis was bilateral arthrosis deformans of TMJ. In patient no. 29, an MRI of the right TMJ showed disk displacement and joint effusion; however, there were no apparent destructive changes of the right condyle head (Fig. 5), and TMJ remodeling was suspected. Cytopathologic Variation in Patients with Similar Background and Clinical Symptoms Patients no. 12, 14, and 17 were females in their 40s with a 20-year medical history of pain and difficulties opening the mouth. These patients had no pre-existing medical condition of mandibular deviation. Although these patients received splint therapy and had similar clinical symptoms with the exception of joint sounds, the cytopathology of the three cases were different. Neutrophils and lymphocytes were predominant in patient no. 12, monocyte lineage cells were found in addition to neutrophils and lymphocytes in patient no. 14, and no cell components were detected in patient no. 17. Patients no. 23 and 24 were both females in their 50s with a five or six month history of pain and/or joint sound. These patients displayed no mandibular deviation and difficulties in mouth opening. Although both patients received splint therapy and had similar clinical symptoms, lymphocytes were detected only in the joint lavage fluid of patient no. 24 and no cell components were detected in that of patient no. 23. Discussion Cytopathologic analyses of 44 samples of joint lavage fluid from 39 TMD patients were carried out. The analyses revealed that cell components, such as neutrophils, lymphocytes, plasma cells, eosinophils, monocyte lineage cells, and cartilaginous tissues, were detected in 50% of the cases. We also observed a correlation between clinical and cytopathologic findings, repeatability of the examinations, and cytopathologic variation in patients with similar symptoms. The synovial fluid of a healthy individual normally contains a small population of different cell types such as monocytes, lymphocytes, and free-floating synovial cells. 32 Traumatic irritations of crepitation, clicking, displacement of the disk, or clenching induce inflammation in the joint. Theoretically, the number of cells detected in the joint lavage fluid should increase in correlation to the level of inflammation. Takano et al. 33 described that the synovial fluid of TMD patients contained a significantly higher number of cells than the fluid Diagnostic Cytopathology, Vol 42, No 1 33

5 MIKAMI ET AL. Fig. 2. Immunocytochemistry of multinucleated giant cells on serial sections from patient no. 14. CD68, alpha 1-antitrypsin, alpha 1-antichymotrypsin were positive and the multinucleated giant cells turned out to be a monocyte lineage cell. (A) HE staining; (B) CD68; (C) alpha 1-antitrypsin; (D) alpha 1-antichymotrypsin; Bar: 50 lm. *Gel used for making cell block. of volunteers who had no symptoms of TMD. Cytopathologic variation seen among TMD patients with similar background and clinical symptoms suggesting that cytopathology of TMJ lavage fluid could provide additional information to what is found from clinical findings and image examinations. In normal cytopathologic diagnosis using smear preparation, it is impossible to perform immunocytochemistry or make serial sections; however, we can use the cell block tissue array method to make paraffin-embedded cell sections, and perform immunostaining on these serial sections. Changes in condylar morphology, as a result of remodeling, is a normal biological adaptation to the continual functional demands made on the TMJ. Differentiating between degenerative changes and adaptive remodeling through image investigations is often difficult. 34 In this study, patient no. 14 was clinically diagnosed as bilateral arthrosis deformans of TMJ because the destructive changes of bilateral condyle were evident from the image investigations, and this was supported by cytopathologic findings of the joint lavage fluid, which showed presence of monocyte lineage multinucleated giant cells (osteoclasts). The detection of monocyte lineage cells from cytopathologic analysis can help determine whether remodeling has taken place, which may not be apparent solely from image investigations. In patient no. 29, the image investigations revealed disk displacement; however, there were no destructive changes of right condyle head. Therefore, remodeling of the condyle inferred based on the presence of monocyte lineage multinucleated giant cells (osteoclasts) in the joint lavage fluid. Crystalline materials are occasionally undetectable with panoramic radiographs, 2 4 and ectopic cartilaginous components are also undetectable in many cases It appears that early stage symptoms of some TMJ diseases are clinically similar to those of TMD and TMJ diseases in the early stage can be misdiagnosed with TMD. 15 Detecting the crystalline components, monocyte lineage cells (macrophages or 34 Diagnostic Cytopathology, Vol 42, No 1

6 CYTOPATHOLOGIC DIAGNOSIS FOR TMD Fig. 3. Fibrous textures were found in the joint lavage fluid in case no. 31. They consisted of cartilaginous small islands with partial calcification and were surrounded by perichondrium. Each island consisted of a few dozen oval-shaped chondrocytes with nuclear pleomorphisms and extracellular matrix. (HE staining; Bar: 100 lm). *Gel used for making cell block. Fig. 5. Magnetic resonance imaging of the right temporomandibular joint in patient no. 29. Disk displacement and joint effusion were seen, however, there were no destructive changes to the right condyle head. Sagittal tomography. Arrow, joint effusion. Fig. 4. Coronal computed tomography of patient no. 14 showed destructive changes of the bilateral condyle. The destructive change was more evident on the right side. foreign body-type giant cells) or cartilaginous components in joint lavage fluids by cell block tissue array method can be helpful for an earlier definitive diagnosis and to avoid invasive treatments, such as arthrotomy or synovectomy. 15 For crystalline arthritis, presence of macrophages or foreign body-type giant cells in the fluid can be seen under a microscope; 1 3,5,8 12 however, detecting the actual crystalline components on the specimens is essential for a definitive diagnosis. These cells usually cannot be detected alone in the tissue or in the aspirated samples from patients with crystalline arthritis. In the samples, crystalline components seem to be more detectable than cell components. In the present study, crystalline materials were not detected in any of the cases. Detailed crystal analysis using X-ray diffraction or electron microscopy are necessary for establishing a definitive diagnosis, but polarizing microscopy can be used as a tool for provisional diagnosis by detecting calcium pyrophosphate dihydrate (CPPD) crystals and monosodium urate (MSU) crystals in the synovial fluid. CPPD crystals show weak positive birefringence, and MSU crystals seen in gout show strong negative birefringence. Since the size of hydroxyapatite crystals is beyond the resolution limits of optical microscopy; they cannot be identified in the synovial fluid. Systemic findings of high blood pressure, medical history of arthritis, or gout are also important for the diagnosis of crystalline arthritis. 12 There are reports of correlations between the presence of inflammatory cytokines (interleukin(il) 1 beta, IL-6, IL-8, IL-10, matrix metalloproteinase(mmp)2, MMP3, MMP9, beta-glucuronidase, IgA, and IgG) in the joint lavage fluid of TMD patients and the clinical outcome Cytokine levels in the joint lavage fluid can also provide detailed information about the inner local condition of the TMJ at the cellular level, or even in gene level. 35 Therefore, examinations of cytopathology with cytokine levels in the joint lavage fluid of a TMD patient could provide more additional information to complement the clinical examinations. In this study, we observed the presence of various kinds of cell components in the TMJ lavage fluids of TMD patients using the cell block tissue array method, and also determined that it was possible to make a distinction between acute inflammation, chronic inflammation, possible remodeling of the TMJ, and synovial chondromatosis. After joint lavage treatments, the joint lavage fluids are routinely disposed. However, our study indicates that cytopathologic examination of the lavage fluids is recommended in order to understand the inner local conditions in the TMJ. Diagnostic Cytopathology, Vol 42, No 1 35

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